1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using regulatory elements from the rice actin 2 gene.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al., 1992), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), and nos (Ebert et al., 1987).
Examples of tissue specific promoters which have been described include the lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al, 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz, 1991), Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990), xcex1-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989) and chalcone synthase promoters (Franken et al., 1991).
Examples of inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Scwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
The rice actin 1 promoter constitutes a particularly useful promoter for expression of transgenes in plants (Wang et al., 1992; U.S. Pat. No. 5,641,876). The rice actin 1 gene, Act1, encode a transcript that is relatively abundant in all rice tissues and at all developmental stages examined. A complete structural analysis of the rice Act1 gene has led to the identification and localization a 5xe2x80x2 intron from the first coding exon of the Act1 sequence (McElroy et al., 1990a). This 5xe2x80x2 intron was found to be essential for the efficient function of the actin 1 promoter.
Plant actin is encoded by a gene family present in all plant species studied to date (Meagher, 1991). In rice, there are at least eight actin-like sequences per haploid genome. Four of the rice actin coding sequences (rice actin 1, 2, 3 and 7) have been isolated and shown to differ from each other in the tissue and stage-specific abundance of their respective transcripts (Reece, 1988; McElroy et al., 1990a; Reece et al., 1990; U.S. Pat. No. 5,641,876; Genbank Accession numbers X15865, X15864, X15862, and X15863, respectively). In situ histochemical localization of the product of a fusion between the actin 1 promoter and a gus reporter gene in transgenic rice plants revealed that the Act1 5xe2x80x2 region is active in most, but not all, sporophytic cell types as well as in gametophytic pollen tissues (McElroy et al., 1990b; 1990c). This pattern is believed to reflect an ubiquitous requirement for cytoskeletal components in plant cells (Zhang et al., 1991).
While the above studies have provided an understanding of the coding regions of rice actin genes, little is still known regarding the 5xe2x80x2 regulatory regions of all but the rice actin 1 gene. In particular, there has been a failure in the art to identify the structure, sequence and function of the rice actin 2 upstream regulatory regions. The identification and sequencing of these regions could potentially provide valuable new tools for the preparation of transgenic plants.
Therefore, according to the present invention, there is provided an isolated rice actin 2 promoter. More particularly, there are provided rice actin 2 promoter sequences isolatable from the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In specific embodiments, there are provided nucleic acid segments of from about 40 to about 743 contiguous nucleotides, from about 60 to about 743 contiguous nucleotides, from about 125 to about 743 contiguous nucleotides, from about 250 to about 743 contiguous nucleotides, from about 400 to about 743 contiguous nucleotides, or from about 600 to about 743 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:2. Alternatively, the isolated nucleic acid may comprise the entire nucleic acid sequence of SEQ ID NO:2.
In a second embodiment of the present invention, there is provided an isolated rice actin 2 intron. More particularly, there are provide rice actin 2 intron isolatable from the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3. In specific embodiments, there are provided nucleic acid segments of from about 40 to about 1763 contiguous nucleotides, from about 100 to about 1763 contiguous nucleotides, from about 300 to about 1763 contiguous nucleotides, from about 700 to about 1763 contiguous nucleotides, from about 1200 to about 1763 contiguous nucleotides, or from about 1500 to about 1763 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:3. Alternatively, the isolated nucleic acid may comprise the entire nucleic acid sequence of SEQ ID NO:3.
Also provided are expression vectors including those where a rice actin 2 promoter is operably linked to a selected gene. It also may be linked to a terminator region. Further, the vector may comprise a genetic element which enhances the expression of said selected gene when said expression vector is stably transformed in the genome of a host plant. The genetic element may be the rice actin 1 intron or the rice actin 2 intron. The expression vector may be a plasmid.
The selected gene may be an insect resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode disease resistance gene, a herbicide resistance gene, a gene affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a negative selectable marker, a gene affecting plant agronomic characteristics, and an environment or stress resistance gene.
The selected gene may be a selectable marker gene encoding a protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase and anthranilate synthase.
The expression vector may further encode a transit peptide, such as the chlorophyll a/b binding protein transit peptide, the small subunit of ribulose bisphosphate carboxylase transit peptide, the EPSPS transit peptide or the dihydrodipocolinic acid synthase transit peptide. The expression vector may encode any of the above-noted selected genes.
Another expression vector of the present invention comprises an isolated rice actin 2 intron. This vector may further comprise a promoter operable in plants and may comprise a terminator. It may comprise a plasmid.
The promoter may be a gamma zein promoter, an oleosin ole16 promoter, a globulin1 promoter, an actin 1 promoter, an actin c1 promoter, a sucrose synthetase promoter, an INOPS promoter, an EMB5 promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter, an Ltp1 promoter, an Ltp2 promoter, an oleosin ole17 promoter, an oleosin ole18 promoter, an actin 2 promoter, a pollen-specific protein promoter, a pollen-specific pectate lyase promoter, an anther-specific protein promoter, an anther-specific gene RTS2 promoter, a pollen-specific gene promoter, a tapetum-specific gene promoter, tapetum-specific gene RAB24 promoter, a anthranilate synthase alpha subunit promoter, an alpha zein promoter, an anthranilate synthase beta subunit promoter, a dihydrodipicolinate synthase promoter, a Thi1 promoter, an alcohol dehydrogenase promoter, a cab binding protein promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme promoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, a regulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, a cellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteine hydrolase promoter, a superoxide dismutase promoter, a C-kinase receptor promoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNA promoter, a glucose-6 phosphate isomerase promoter, a pyrophosphate-fructose 6-phosphate1phosphotransferase promoter, an ubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa photosystem II promoter, an oxygen evolving protein promoter, a 69 kDa vacuolar ATPase subunit promoter, a metallothionein-like protein promoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA- and ripening-inducible-like protein promoter, a phenylalanine ammonia lyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteine hydrolase promoter, an xcex1-tubulin promoter, a cab promoter, a PEPCase promoter, an R gene promoter, a lectin promoter, a light harvesting complex promoter, a heat shock protein promoter, a chalcone synthase promoter, a zein promoter, a globulin-1 promoter, an ABA promoter, an auxin-binding protein promoter, a UDP glucose flavonoid glycosy1-transferase gene promoter, an MPI promoter, an actin promoter, an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35S promoter, a CaMV 19S promoter, a histone promoter, a turgor-inducible promoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmid mannopine synthase promoter, Ti plasmid nopaline synthase promoter, a petunia chalcone isomerase promoter, a bean glycine rich protein 1 promoter, a CaMV 35S transcript promoter, a Potato patatin promoter, or a S-E9 small subunit RuBP carboxylase promoter.
In another embodiment of the present invention, there is provided a fertile transgenic plant stably transformed with a selected DNA comprising an actin 2 promoter, and optionally a terminator. The fertile transgenic plant may further comprise a selected gene operably linked to the rice actin 2 promoter. Again, the selected gene may be any of the aforementioned genes. The selected DNA may further comprises a genetic element which enhances the expression of said selected gene in said fertile transgenic plant, such as the rice actin 1 intron and rice actin 2 intron. The selected DNA may comprise a transit peptide, including any of those mentioned above. The plant may be a monocotyledonous plant, such as wheat, maize, rye, rice, turfgrass, sorghum, millet and sugarcane. The plant may be a dicotyledonous plant, such as tobacco, tomato, potato, soybean, sunflower, alfalfa, canola and cotton.
In still another embodiment, there is provided a crossed fertile transgenic plant prepared according to the method comprising the steps of (i) obtaining a fertile transgenic plant comprising a selected DNA comprising an actin 2 promoter; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant comprising said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant. The plant may be a monocot or a dicot as set out above. In a particular embodiment, the plant is maize.
The crossed fertile transgenic plant may have the selected DNA inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. The crossed fertile transgenic may have the actin 2 promoter isolatable from the nucleic acid sequence of SEQ ID NO:2, including those particular segments set forth above. The crossed fertile transgenic plant also may have, as part of the selected DNA, an exogenous gene operably linked to said actin 2 promoter, including any of those set out above. Also included within the present invention are seeds of any of these crossed fertile transgenic plants.
In yet another embodiment, there is provided a crossed fertile transgenic plant prepared according to the method comprising (i) obtaining a fertile transgenic plant comprising a selected DNA comprising an actin 2 intron; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare seed of a crossed fertile transgenic plant comprising said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant comprising said selected DNA. The crossed fertile transgenic plant may be monocot or dicot, as set out above, and in particular is maize.
The selected DNA may be inherited through a female parent or through a male parent. The second plant may be an inbred plant and the crossed fertile transgenic plant may be a hybrid. The actin 2 intron may be isolated from the nucleic acid sequence of SEQ ID NO:3. The selected DNA may further comprises an exogenous gene, including any of those set out above. Also included within the invention are seeds of these plants.
In still yet another embodiment, there is provided a method of expressing an exogenous gene in a plant comprising the steps of (i) preparing a construct comprising said exogenous gene operably linked to an actin 2 promoter; (ii) transforming a recipient plant cell with said construct; and (iii) regenerating a transgenic plant expressing said exogenous gene from said recipient cell. The recipient plant cell may be from a monocot or dicot, particularly maize. The exogenous gene may be any of those set out above.
In still yet a further embodiment, there is provided a method of expressing an exogenous gene in a plant comprising the steps of (i) preparing a construct comprising an actin 2 intron and an exogenous gene; (ii) transforming a recipient plant cell with said construct; and (iii) regenerating a transgenic plant expressing said exogenous gene from said recipient cell. The plant may be monocot, dicot, and in particular is maize. The construct may comprise an exogenous gene including those disclosed above, and the promoter includes any of those disclosed above.
In an additional embodiment, there is provided a method of plant breeding comprising the steps of (i) obtaining a transgenic plant comprising a selected DNA comprising an actin 2 promoter; and (ii) crossing said transgenic plant with itself or a second plant. The second plant may be an inbred plant, a monocot, a dicot, and in particular is maize.
In still a further embodiment, there is provided a method of plant breeding comprising the steps of (i) obtaining a transgenic plant comprising a selected DNA comprising an actin 2 intron; and (ii) crossing said transgenic plant with itself or a second plant. The second plant may be an inbred plant, may be monocot or dicot, and in particular is a maize plant.